PROJECT SUMMARY Viral replicons, or self-replicating RNA elements incapable of initiating a spreading infection, are traditionally packaged into virus particles, termed viral replicon particles (VRPs), for in vivo delivery. Alternatively, they can be encoded on a plasmid DNA and transfected for host cell nucleus-mediated launch of replicating RNA. However, concerns of anti-vector immunity with respect to the former, or genome integration in the latter approach has limited the clinical use of such technologies. Furthermore, the development of VRPs has been restricted to larger viruses that are capable of harboring additional genetic cargo. With improvements in the delivery of naked RNA, there are now opportunities to develop new viral replicons without the constraints provided by traditional delivery paradigms. By utilizing replicons derived from diverse positive- strand RNA virus families, we can exploit the various mechanisms used by the host to detect replicating viral RNA as well as the differential mechanisms of immune evasion utilized by the parental virus, to rationally design vaccines. We propose studies that will elucidate the minimal genetic elements derived from a small positive-stranded RNA animal virus that are required to replicate and express a heterologous gene from a subgenomic transcript. We will then demonstrate the translational applications towards vaccine development for Zika and tuberculosis, viral and bacterial diseases with significant public health consequences. Furthermore, we will perform comparative studies between this platform with existing platform technologies derived from other positive-strand RNA virus families with the hypothesis that each will elicit distinct innate and adaptive immune responses due to evolutionary differences of the parental viruses.